Evaluation of various pretreatment conditions and the effect on key variables, such as the overall yield of sugars or ethanol, needs to be assessed in an easy way to be able to judge the result. In several studies on pretreatment of biomass the “severity factor” has been used for comparing pretreatment results. Although it does not provide an accurate measure of the severity it can be used for rough estimates [8,9]. The severity correlation describes the severity of the pretreatment as a function of treatment time (minutes) and temperature (°C), Tref = 100 °C.

log(Ro) = log (t exp ^ ^ef) j j. (1)

When pretreatment is performed under acidic conditions (e. g. by impregna­tion with H2SO4), the effect of pH can be taken into consideration by the combined severity [10] defined as:

Combined severity (CS) = log(Ro) — pH (2)

It is well known that more severe conditions during pretreatment will cause greater degradation of hemicellulose sugars [11-13]. A high severity in the pretreatment is nevertheless required to enhance the enzymatic digestibility of cellulose [14]. The ideal pretreatment would hydrolyse the hemicellulose to its monomer sugars without further degradation. It would also cause an increase of the pore size and reduce cellulose crystallinity to enhance the en­zymatic digestibility of the cellulose fibres. However, these two effects are not reached at the same pretreatment severity, at least not using current technologies.

Assessment of pretreatment is usually done by using some of (or a combi­nation of) the following methods:

1. Analysis of the content of sugars liberated during pretreatment to the liquid as a combination of monomer and oligomer sugars, as well as an­alysis of the carbohydrate content of the water-insoluble solids (WIS). This gives the total recovery of carbohydrates in the pretreatment step.

2. Enzymatic hydrolysis (EH) of the WIS, either washed or non-washed.

3. Fermentation of the pretreatment liquid to assess inhibition of the fer­menting microorganism.

4. Simultaneous saccharification and fermentation (SSF) of either the whole slurry or the washed WIS.

The enzymatic hydrolysis (in 1 and 4) is performed using cellulases, i. e. a mixture of various cellobiohydrolases and endoglucanases supplemented with yd-glucosidase. The latter is not a cellulase as it only cleaves cellobiose into two glucose molecules. It has, however, a very important role in hydro­lysis since cellobiose is an end-product inhibitor of many cellulases [15,16]. On the other hand, d-glucosidase is also inhibited by glucose [17]. Since the enzymes are inhibited by the end products, the build-up of any of these products affects the cellulose hydrolysis negatively. The maximum cellulase activity for most fungus-derived cellulases and d-glucosidase occurs at 50 ± 5 °C and a pH of 4.0-5.0. However, the optimal conditions for enzymatic hydrolysis change with the hydrolysis residence time [18] and are also depen­dent on the source of the enzymes.

The enzymatic hydrolysis for assessment of pretreatment can be per­formed using various conditions (substrate concentration, enzyme dosage, temperature, stirring speed etc.). A common way is to use washed material at 2 wt % WIS, or alternatively at 1 wt % cellulose, to avoid end-product in­hibition [19]. This could be seen as the maximum achievable digestibility or glucose yield. However, it does not reflect the pretreatment efficiency in terms of avoiding formation of compounds that are inhibitory to the cellulases. In a full-scale process it is crucial to reach high sugar and ethanol concentrations in order to decrease the energy demand in the downstream processes. To in­crease the sugar concentration during large-scale operation, it is assumed that the whole slurry after pretreatment would be used without introducing sepa­ration steps, which would dilute the process stream. Furthermore, the overall substrate loading in enzymatic hydrolysis would probably need to be above 10 wt % WIS to meet the energy requirement for ethanol recovery. To mimic a situation that will be more similar to final process conditions, the enzymatic hydrolysis can be performed using the whole slurry from the pretreatment di­luted to various WIS concentrations, e. g. 10 wt %. In this case also the effect of inhibitors is assessed. However, due to the higher concentration of sugars the enzymes will also suffer from end-product inhibition.

To assess the effect of possible inhibitors acting on the microorganism used for fermentation of the sugars released in the enzymatic hydrolysis, method 2 is most often combined with method 3. The overall ethanol yield depends not only on the sugar yield, but also on the fermentability of the solution. Inhibition is influenced by the concentration of the soluble sub­stances released during pretreatment, present in the original raw material, e. g. acetic acid, or formed in the pretreatment step. Some of the substances present in the slurry are furfural and 5-hydroxymethylfurfural (HMF), which are the result of degradation of pentoses and hexoses, respectively. Furfural may react further to yield formic acid, or it may polymerize. HMF can be converted to formic acid and levulinic acid. In some pretreatments lignin degradation products are also formed. The concentrations of these and all other inhibitory substances in the fermentation step depend on the con­figuration of the preceding process steps. At ethanol concentrations below 4 to 5 wt % the energy demand increases rapidly with decreasing ethanol concentration. It is thus important to evaluate the fermentability of the con­centrated pretreatment hydrolysates. The fermentability test is usually per­formed on the liquid obtained from the pretreatment, either directly or di­luted to a concentration corresponding to what is expected to be suitable in a final process.

Another option for evaluation of the pretreatment step is to perform SSF either on the whole slurry diluted to a suitable WIS concentration or on the washed water-insoluble solid material, in both cases at a WIS around 5% or higher. In this case the glucose produced is immediately consumed by the fermenting microorganism, e. g. Saccharomyces cerevisiae, which removes the end-product inhibition of glucose and cellobiose. SSF adds information about the pretreatment efficiency, since SSF usually gives a higher overall ethanol yield than separate enzymatic hydrolysis and fermentation (SHF) due to con­version by the microorganism of some compounds that are inhibitory to the enzymes to less inhibitory compounds [20]. Also in the assessment by SSF the conditions may vary, e. g. substrate concentration, enzyme dosage, concentra­tion of microorganism etc.

It has to be added that variations between different laboratories in con­figurations and conditions used for assessment of the pretreatment make it very difficult to compare various pretreatment methods unless they are assessed in exactly the same way. Even so, the conclusions may be incor­rect as the conditions used may be unfavourable to a specific method. For instance, the use of hemicellulases in the enzymatic hydrolysis, instead of only cellulases, will be beneficial to pretreatment methods that result in large amounts of oligomer hemicellulose sugars, as will be discussed in the results section.

It is our opinion that the assessment of pretreatment has to be performed in a more rigorous way. The standard enzymatic hydrolysis at low substrate concentration may well be used to assess the maximum digestibility. However, in this case both cellulases and hemicellulases are needed. The “real” assess­ment should be performed by optimizing the conditions for all subsequent process steps under more realistic process conditions, taking into account the special features of the pretreated material, and then comparing the produc­tion cost for the various alternatives.

3

The primary factor in the high cost of enzymes for biomass hydrolysis is sim­ply the amount of enzyme that must be used. Compared to starch hydrolysis, 40- to 100-fold more enzyme protein is required to produce an equivalent amount of ethanol (Novozymes data). It was recognized very early on that efficient cellulose hydrolysis requires a complex, interacting collection of en­zymes during initial characterization of the T. reesei cellulase system [35]. To significantly reduce the amount of these enzymes requires that either more efficient component enzymes are identified or that additional enzymes can be added that reduce the total enzyme loading. Synergy, the ability of two or more enzymes to work simultaneously more effectively than in succession, was first described in cellulases more than 30 years ago when describing the action of CBH I and EG activities [36]. In this case, the synergy can be mech­anistically explained by the production of new cellulose ends by the action of the endoglucanase, creating new sites of exoglucanase attack by the CBH. Similarly, studies of the observed synergism between CBH I and CBH II from Humicola insolens, revealed that this CBH II, although capable of acting pro — cessively from non-reducing chain ends, does also cleave the cellulose chains in an endo fashion [37]. To drive enzyme loading down, we needed to search for similar synergistic enzyme pairs that could complement the preferred T. reesei cellulase system.

4.2.1

Pretreatment strategies have generally been categorized into biological, phys­ical and chemical processes, or a combination of these approaches.

Biological pretreatments typically utilize wood degrading fungi (soft, brown and white rot) to modify the chemical composition of the lignocel — lulosic feedstock. Generally, soft and brown rot fungi primarily degrade the hemicellulose while imparting minor modifications to lignin. White-rot fungi can more actively attack the lignin component [12]. Although there has been a fair amount of work done in this area, the primary application has been as a biopulping option for the pulp and paper industry rather than as a pretreat­ment for bioenergy applications. In addition to the requirements for careful control of growth conditions and for large amounts of space to perform bio­logical treatments, a major disadvantage of biological/fungal treatments is the typical residence time of 10-14 days. For these reasons, biological pretreat­ments are considered to be less attractive commercially.

Physical pretreatments involve the breakdown of the biomass feedstock into smaller particles that are more amenable to subsequent enzymatic hydrolysis. Physical treatments such as hammer — and ball-milling [13-16] have been shown to improve hydrolysis yields by disrupting cellulose crys­tallinity and by increasing the specific surface area of the feedstock, rendering them more accessible to attack by cellulases. One of the advantages of physi­cal pretreatment is that it is relatively insensitive to the physical and chemical characteristics of the biomass employed. However, the physical pretreatment processes are energetically demanding and do not result in lignin removal. Lignin has been shown to restrict access and inhibit cellulases [17,18]. Fur­thermore, physical pretreatments have yet to be shown to be economically viable at a commercial scale.

Most of the chemical pretreatments that have been assessed to date (typ­ically acid and alkali based) have had the primary goal of enhancing enzyme accessibility to the cellulose by solubilizing the hemicellulose and lignin, and to a lesser degree decreasing the DP and crystallinity of the cellulosic component. Pretreatments that reduce cellulose crystallinity include mild swelling agents such as NaOH, hydrazine and anhydrous ammonia, and ex­treme swelling agents such as sulfuric acid, hydrochloric acids, cupram, cuen, and cadoxen [19]. Treatments that reduce the lignin content of the substrate include organosolv pulping with various solvents including ethanol, glycerol and ethylene glycol.

Typically, all chemical pulping processes in commercial use today involve the removal of lignin to produce pulp for various paper products. Although these processes could be considered as potential pretreatment methods, they are optimized to maintain the fiber/strength integrity of the pulp, not to in­crease accessibility to the cellulose. The relatively high value of pulp (at the time of writing, approximately US$730 per tonne of northern bleached soft­wood Kraft pulp in Europe according to the PIX Pulp Benchmark Index) can justify the high capital and operating costs of chemical pulping, while lower-value biofuels must seek cheaper pretreatment alternatives. Despite these apparent drawbacks, various groups have looked at modified pulping processes as potential pretreatment methods, most likely since these pulp­ing processes produce readily hydrolyzable substrates. For example, in a Kraft pulping process NaOH and Na2S are combined in an aqueous liquor to cook wood chips under elevated pressures, followed by a pressure-release defi — bration step. The resulting Kraft pulps have been shown to be receptive to subsequent hydrolysis by cellulases [16], most likely because of the combina­tion of chemical dissolution of lignin and a decrease in average particle size that occurs during physical defibration.

Pretreatments that combine both chemical and physical processes are re­ferred to as physiochemical processes. These pretreatment methods have received the most attention in recent years and are the major focus of this review. In particular, steam pretreatment has received significant attention for its suitability in generating easily hydrolyzable substrates from lignocellu — losic biomass. However, several aspects that affect the viability of the overall process will be discussed in more detail later in this review, including the handling and preparation of the feedstock prior to the pretreatment step, the need to minimize processing costs, and the need to maximize the value of co­products derived from the hemicellulose and lignin streams. For example, if a pretreatment method has a requirement for very fine, uniform feedstock with a particle size of less than 10 mm, this will have a significant impact on the overall economic viability of the overall process because of the en­ergy requirements to produce this fine material [20,21]. Similarly, although acid-based pretreatment processes have been shown to be effective on a range of lignocellulosic substrates, downstream costs including the need for alka­line neutralization chemicals such as CaOH2 [22], must be considered. At the same time alkaline-based pretreatment methods such as lime, ammonia freeze explosion (AFEX), and ammonia recycle percolation (ARP) processes can effectively reduce the lignin content of agricultural crops such as wheat straw and corn stover, but have a much more difficult time processing recal­citrant substrates such as softwoods.

To summarize this general introduction, it is unlikely that one pretreat­ment process will be declared a “winner” as each method has its inherent advantages/disadvantages. However, as discussed in more detail below, steam pretreatment is one method that is effective on a range of lignocellulosic sub­strates and, through companies such as Masonite, has been shown to work effectively at a commercial scale.

1.2

During the same time period that the cost of crude petroleum rose 150%, from January 2001 to 2005, the total number of bioethanol refineries in the USA increased from 56 to 81, with total production capacity increasing from 6.6 bil­lion L/year to 13.8 billion L/year. Within the last year, from January 2005 to 2006, the total number of refineries increased to 95 and output further increased to 14.3 billion L/year, a 1500% increase since 2001. Total world production in 2005 was 46 billion L, with the USA and Brazil representing a combined 70% of the world’s production. It should be further noted that by the end of 2005, 29 ethanol refineries and nine expansions of existing refineries were under con­struction, with a combined annual capacity of 5.7 billion L. If you consider all of the US ethanol production capacity currently on-line, under expansion, and under construction, then the projected capacity is approximately 24 billion L — approximately 85% of that required by the RFS by 2012 [43].

In the USA, the raw material of choice for bioethanol production is corn. Approximately 13% of the US corn crop is dedicated to ethanol production,

third only to livestock feed and exports [43]. In Brazil, however, the raw ma­terial of choice is sugarcane. With over 100 countries producing sugarcane, no one has yet to match Brazil’s cost structure and supply chain. In mid-2005, the sugar production costs in the three lowest countries were estimated to be $145/metric ton in Brazil, $185/metric ton in Australia, and $195/metric ton in Thailand. About 25% of worldwide sugar production is at $200-250/metric ton, above which the figure escalates to $400/metric ton and higher. Sugar­cane is a highly efficient crop for producing biomass, representing the highest biomass per growing area of any major commercial crop, including corn. This is a result of sugarcane’s ability to incorporate C3 and C4 compounds in its photosynthetic pathway, while most plants only incorporate C3 compounds. Brazilian ethanol is most likely the cheapest in the world, with an estimated production cost of between $0.19 and $0.21/L in 2005. For this reason Brazil is not only looking to expand its ethanol production capacity, but to further expand into biorefineries [54].

Brazil’s sugarcane production is unique, and not representative of the gen­eral challenge almost all other nations face when determining which raw ma­terial source is preferred. Raw material utilization for bioethanol and biotech­nology processes in general represents a significant challenge and opportu­nity for research and development. The US Department of Agriculture and Department of Energy estimate that the resources exist to produce over 1 bil­lion tonnes of biomass annually, representing approximately 30% displace­ment of current fossil fuel usage (302 billion L) [55]. Biomass is composed of cellulose (40-50%), hemicellulose (25-35%) and lignin (15-20%) [56]. Significant effort in the fields of non-food agricultural engineering, enzyme catalysis of cellulose and hemicellulose, and hexose and pentose fermentation will be required to extrapolate the full energetic value of lignocellulose.

Figure 2 schematically shows how research in the aforementioned areas is integrated into bioethanol process development, specifically focusing on the secondary pretreatment of feedstocks and microbial metabolic engineer­ing. In both examples, the application of systems biology to the metabolic engineering framework can yield improved products, either in the form of enzymes or microbial platforms. We will further explore how scientific and technical achievements in the fields of metabolic engineering and systems bi­ology as applied to the afore mentioned areas and others, driven by industrial biotechnology and demand for bioethanol, will improve bioethanol process development.

3

There have been only limited studies assessing the significance of initial cellulose crystallinity and DP of lignocellulosic substrates with regard to subsequent substrate hydrolysis by cellulases; however, the importance of these factors has been the subject of considerable debate [113,114]. It has been suggested that amorphous cellulose is hydrolyzed, initially resulting in an accumulation of crystalline cellulose rendering the substrate increasingly recalcitrant as the hydrolysis progresses [113,114]. Most studies that have established a correlation between crystallinity and hydrolysis have utilized substrates of relatively pure cellulose, which most likely do not represent the heterogeneous lignocellulosic substrate encountered during the hydrolysis of substrates pretreated for bioconversion [115-117]. Furthermore, to demon­strate the effect of crystallinity on hydrolysis, these studies frequently utilize physical treatments such as ball milling [116] or gamma irradiation [118] to alter the initial substrate crystallinity, which can also result in increases in specific surface area. As a result, in previous work both crystallinity and spe­cific surface area of pure cellulose substrates have been combined into models predicting the rate and extent of hydrolysis [119]. As for crystallinity, it is dif­ficult to assess the effects of DP exclusively, since altered DP can be associated with crystallinity or accessible surface area. Nevertheless, there have been a few studies investigating the effects of the crystallinity and DP of chemical pulps on their hydrolysis by cellulases.

Employing unbeaten, beaten and recycled softwood pulps as substrates to assess various substrate characteristics that influence the enzymatic hydro­lysis of cellulose, Nahzad et al. [105] showed that although the pulps pos­sessed similar crystallinity, the beaten pulp hydrolyzed more readily than the unbeaten pulp without any appreciable changes in crystallinity occur­ring during the hydrolysis. Similar results have been found by Ramos et al. during the treatment of fully bleached eucalyptus Kraft pulp [104] and by Mansfield et al. during combined cellulase-xylanase treatment of Douglas — fir Kraft pulps [99]. Nahzad et al. [105] also showed that the initial DP of the pulps did not play a role in affecting subsequent hydrolysis; however, the DP was decreased by 2/3 during the hydrolysis period and the polydispersi — ties of all the hydrolyzed pulps were quite similar. Mansfield et al. [99] did not observe appreciable changes in cellulose DP during hydrolysis, however, it should be noted that they employed low cellulase loadings to impart sub­tle modifications to the pulp fiber. It is evident from the literature presented here that attributing the ease of enzymatic digestibility of a given substrate to initial crystallinity or DP is a dubious task, compared to studies that have tied the ease of hydrolysis of substrates to their initial surface area. However, pore volume determinations require a significant investment in time to ob­tain reproducible results. Also, it is likely that the pores in a lignocellulosic substrate will have irregular shapes, thus affecting the accuracy and preci­sion of the measurement [120]. Another drawback is that the method does not measure the areas in pores that are larger than the size of the probe, which would provide the easiest access for cellulases. Investigations into the sub­strate physical factors that affect hydrolysis should be aided by the continual evolution of analytical techniques such as thermoporosimetry [121] and high resolution fiber quality analysis [122], which may be capable of dealing with the diversity of pretreated lignocellulosic substrates produced for subsequent hydrolysis and fermentation in the bioconversion process.

6

Conclusions

In this review we suggested that, although the properties of the cellulase en­zyme complex has a significant effect on how effectively a lignocellulosic material will be hydrolyzed, it is the biomass pretreatment and the intrin­sic structure/composition of the substrate itself that are primarily responsible for its subsequent hydrolysis by cellulases. It is apparent that in sequential series of events, the conditions employed in the chosen pretreatment will af­fect various substrate characteristics, which in turn govern the susceptibility of the substrate to hydrolysis by cellulases and the subsequent fermentation of the released sugars. Choosing the appropriate pretreatment for a particu­lar biomass feedstock is frequently a compromise between minimizing the degradation of the hemicellulose and cellulose components while maximizing the ease of hydrolysis of the cellulosic substrate. The digestibility of pretreated lignocellulosic substrates is further complicated by the lignin-hemicellulose matrix in which cellulose is tightly embedded. Pretreatment conditions can be tailored to create either solid or solid/liquid substrates with varying levels of cellulose, hemicellulose and lignin. It is apparent that lignin affects enzy­matic hydrolysis by blocking cellulose and by chemical interactions facilitated by its hydrophobic surface properties and various functional groups. The role of hemicellulose is less obvious although there is good evidence to support the action of hemicellulose as a barrier restricting access to cellulases. In the past, many investigators have attributed the enhanced enzymatic hydrolysis performance of a particular pretreatment to changes in the proportion of the lignin, hemicellulose and cellulose in the substrate. However, it is important to advance this conclusion one step further as it is likely that decreases in lignin and hemicellulose content that occur as a result of pretreatment also affect the physical properties of the cellulosic component, such as its crys­tallinity, the degree of polymerization and the surface area of the substrate accessible to cellulases.

Various studies conducted with different cellulase systems and a range of cellulosic substrates all indicate that it is ultimately the “accessibility” of the cellulose fraction to the enzyme system that determines how fast (reaction rate) and how far (% conversion) the hydrolysis reaction can proceed [112]. In work conducted with either wood pulps or substrates pretreated for bio­conversion we and other groups have shown that accessibility is a property that describes the static environment encountered by the cellulase complex when it is combined with the substrate, and its action is governed by the in­trinsic pore size distribution, degree of swelling and other gross and detailed substrate characteristics. As enzymatic hydrolysis commences, the situation becomes dynamic, as substrate attributes begin to change due to cellulose hydrolysis and the hydrolysis rate decreases. Some workers have reported de­creases in accessible surface area as hydrolysis proceeds without appreciable changes in crystallinity [99,105,123], while others have reported decreases in crystallinity and increases in accessible surface area during hydrolysis [124]. The discrepancy in results regarding the decrease in the hydrolysis rate of pretreated substrates can most likely be attributed to variations in the sub­strates being studied and the techniques used for measurement of substrate properties. Furthermore, as cellulose is hydrolyzed, the lignin and hemicellu — lose that accumulate in the hydrolysis residue can potentially restrict access to cellulases and decrease the hydrolysis rate. Therefore, pretreatments should aim to produce a readily hydrolyzable substrate by increasing accessibility to cellulases and limiting the negative effects of hemicellulose and lignin on hydrolysis, while maximizing the total carbohydrate recovery.

However, it is important to recognize that studies which try to optimize pretreatment (as assessed by product recovery) need to be performed in parallel with measurements of key substrate characteristics in order to as­sociate specific aspects of pretreatment to substrate attributes that facilitate subsequent hydrolysis by cellulases. This emphasizes the significance of the pretreatment since the effectiveness of pretreatment affects both the up­stream selection of biomass, the efficiency of recovery of the overall cellulose, hemicellulose and lignin components, and the chemical and morphological characteristics of the resulting cellulosic component, which governs down­stream hydrolysis and fermentation.

Adv Biochem Engin/Biotechnol (2007) 108: 95-120 DOI 10.1007/10_2007_066 © Springer-Verlag Berlin Heidelberg Published online: 27 June 2007

Progress and Challenges

A multitude of different pretreatment methods have been suggested during the past few decades. They can loosely be divided into different categories: physical (e. g. milling, grinding and irradiation), chemical (e. g. alkali, dilute acid, oxidizing agents and organic solvents), physicochemical (e. g. steam pre — treatment/autohydrolysis, hydrothermolysis and wet oxidation) and biologi­cal, or combinations of these. In general, it is difficult to place the methods into one category only.

A rough classification of the pretreatment methods can also be made ac­cording to the following:

• Acid-based methods, i. e. pretreatment at low pH, result in hydrolysis of the hemicellulose to monomer sugars and minimize the need for hemicel — lulases.

• Methods working close to neutral conditions, e. g. steam pretreatment and hydrothermolysis, solubilize most of the hemicellulose due to the acids re­leased from the hemicellulose, e. g. acetic acid, but do not usually result in total conversion to monomer sugars. This thus requires hemicellulases acting on soluble oligomer fractions of the hemicellulose.

• Alkaline methods leave a part of the hemicellulose, or in the case of ammonia fibre explosion (AFEX), almost all hemicellulose in the solid fraction. This then requires hemicellulases acting both on solid and on dissolved hemicellulose. An alternative is to perform an acid hydrolysis of this fraction, for instance after removal of the cellulose by enzymatic hydrolysis.

This affects, of course, not only the method that should be used for assess­ment of the pretreatment but also the cost of the overall hydrolysis of the carbohydrates.

3.1

An “efficient” cellulase system requires sufficient в-glucosidase (BG) to hy­drolyze cellobiose produced by the action of the CBHs to prevent their prod­uct inhibition [38]. The addition of BG to a complex cellulase mix such as the Novozymes Celluclast 1.5 L dramatically improves the extent and, during the later stages of hydrolysis, the rate of cellulose saccharification. This is re-

flected in Fig. 6, where the T. reesei strain used to produce Celluclast 1.5 L was compared to the same strain expressing Aspergillus oryzae BG in hydro­lysis assays. Due to relief of the product inhibition at high solids loadings (13.5% w/w in this example), the amount of total enzyme protein required to hydrolyze 80% of the cellulose to glucose was reduced by nearly twofold. At this solids loading, the beneficial effect of BG addition was saturated when it reached ~ 5% of the total enzyme protein, but higher solids would require higher BG levels or a more active BG.

4.2.2

Over the past 20 years, our group has looked at steam pretreatment (SP) with regard to its suitability for pretreating a range of lignocellulosic sub­strates, the subsequent ease of enzymatic hydrolysis of the cellulosic stream and the recovery of most of the hemicellulose sugars and lignin in a use­able form. SP is an attractive pretreatment process as it makes limited use of chemicals, requires relatively low levels of energy and, depending on the conditions employed, results in the recovery of most of the original cellulose — and hemicellulose-derived carbohydrates in a fermentable form [23-28]. As will be discussed in more detail, SP disrupts the lignin barrier [29] and facili­tates access of cellulases to the cellulose fibers [30]. Previous work has shown that SO2-catalyzed SP is an effective pretreatment for softwood [26-34], hard­wood [35-37] and agricultural residues [22] and that impregnation of SO2 prior to pretreatment results in lower treatment temperatures and shorter reaction times, thereby improving hemicellulose recovery and reducing the
formation of sugar degradation products [23]. It has also been shown that SO2 impregnation prior to SP enhanced the carbohydrate hydrolysis rate by increasing the accessibility of cell walls via the formation of fractures and the removal of hemicellulose during the steaming of the substrate [28], while re­ducing the DP of the oligomers and increasing the proportion of monomers

♦

in the water-soluble stream [31,38-40]. The “severity” of SP can be summa­rized by a single factor called Ro (Ro = t exp(T — 100)/14.75) which links the effects of time (t, min) and temperature (T, °C) [41]. Due to the high tem­perature and acidic conditions employed during the SP process, depending on the “severity” (temperature, time, pressure, catalyst dosage) of the treat­ment, a portion of the hemicellulose-derived sugars and solubilized lignin fragments can be degraded or transformed into compounds such as furfural and 5-hydroxymethylfurfural (5-HMF); aliphatic acids, such as acetic, formic, and levulinic acid; and phenolic compounds [42]. It is known that these compounds can inhibit both downstream hydrolysis by cellulases [43] and fermentation of the liberated sugars to ethanol [44]. Therefore, compromised SP conditions have to be defined that provide an easily hydrolyzable cellu — losic substrate, good recovery of the hemicellulose-derived sugars, ideally in a monomeric form, while minimizing the formation of inhibitors. Ideally, a reactive lignin stream, with a higher economic application than its intrinsic fuel value should also be obtained.

It is apparent that the nature of the substrate and the pretreatment method used has at least as much influence on the ease of enzymatic hydrolysis as does the nature and efficiency of the enzyme system used to conduct hydrolysis. As illustrated in Fig. 1, the efficiency of enzymatic hydrolysis of a given lignocellu — losic substrate is the result of interplay of various factors. Although it is evident that substrates such as agricultural residues are generally less recalcitrant than softwood residues, it is recognized that enzyme — or substrate-related factors that govern effective hydrolysis can be controlled to an appreciable extent by the type and conditions of the pretreatment employed.

In the sections below we will describe how pretreatment, specifically SP, in­fluences the characteristics of the substrate and the subsequent recovery of the cellulose, hemicellulose and lignin components. Recent progress in eluci­dating the role of substrate properties such as crystallinity, DP, pore volume, and available surface area in enzymatic hydrolysis will also be discussed using wood pulps as “model substrates”. The final section offers concluding re­marks and outlines the remaining challenges associated with understanding the progress of enzymatic hydrolysis during the bioconversion process.

2

Systems biology is the quantitative characterization of genetic, transcription, protein, metabolic, signaling and other informational pathway responses to
a clearly defined perturbation of a biological system. More specifically, the perturbation may take the form of a genetic, biochemical, or environmental stimulus. At the core of systems biology is the transformation of quantita­tive, typically large-scale data sets, into in silico models that provide both interpretation and prediction. Systems biology has emerged as a tool applied in different fields, including metabolic engineering, to what many consider to be an independent discipline of study and research [57]. Table 1 provides an overview of commonly used industrial biotechnology strategies, focused on metabolic engineering with specific examples taken from applications

Table 1 Overview of commonly used industrial biotechnology strategies

Industrial biotechnology strategy Examples of application to

bioethanol production

A case study considering the co-production of ethanol and succinic acid suggests significant cost reduction (sales price of ethanol decreases from $0.51 to $0.42/gal.). Pilot plant confirmation pending [115,116].

In silico aided metabolic engineering of S. cerevisiae lead to a 40% reduction in glycerol formation and 3% increase in ethanol yield in vivo [154].

Natural ethanol producing bacterium Zymomonas mobilis metabolically engineered to ferment xylose and arabinose as preferred carbon sources via introduction/expression of E. coli pathway genes [6,155].

Xylose (C5H10O5, significant fraction of lignocelluloses) utilization by S. cerevisiae investigated and optimized via introduction of a Piromyces sp. xylose isomerase (XylA). Further xylose metabolic structural genes were overexpressed. Xylose consumption of

0.

9-1.1 g g-biomass-1 h-1, demonstrated in vivo [156-159].

to bioethanol production. The examples cited exploit toolboxes developed within systems biology.

Therefore, we refer here to industrial systems biology, defined as the appli­cation of experimental or numerical methods developed from systems biol­ogy to improve bioprocess development in terms of final product titer, yield, or productivity, or process robustness and efficiency. In most cases, indus­trial systems biology has been product — or process-specific; however, there are emerging examples of successful commercialization of stand-alone systems biology tools and products for broad application [58].

Recent advances in high-throughput experimental techniques have re­sulted in rapid accumulation of a wide range of x-omics data of various forms (Fig. 3), providing a foundation for in-depth understanding of biolog­ical processes [59-62]. How to integrate, interpret, and apply these data is an area of active research. Bioinformatics has become a well-established and recognized interdisciplinary field. To date, large data sets of transcriptomes, metabolomes, and to lesser degrees proteomes and fluxomes, for multiple organisms have been acquired. Resources are being applied to integrating the various data sets for in silico simulations and creating relevant models that represent in vivo physiological conditions of host cells responding to environmental stimuli. Even though our ability to analyze these x-omic (see “Glossary”) data in a truly integrated manner is limited, new targets for strain improvement can be identified from these global data [63-69].

A sampling of recent developments and applications in the field of sys­tems biology will be discussed in relation to improving the productivity of bioethanol. Examples will be provided on single x-ome approaches and com­bined analysis of these x-ome data for the development of improved strains and enhancement of metabolic engineering strategies.

3.1

Sandra T. Merino • Joel Cherry (И)

Novozymes Inc., 1445 Drew Ave., Davis, CA 95618, USA JRoC@novozymes. com

1 Introduction……………………………………………………………………………………………….. 96

2 Lignocellulosic Biomass to Ethanol Process Overview……………………………………. 97

2.1 Minimizing Yield Loss and Cost…………………………………………………………………… 99

3 Impact of Process Steps on Enzyme Dosage and Cost…………………………………. 100

3.1 Impact of Substrate Selection on Enzyme Cost……………………………………………. 101

3.2 Impact of Pretreatment Selection……………………………………………………………….. 102

3.3 The Impact of Process Integration on Enzyme Requirements………………………… 104

4 Enzyme Discovery: Catalytic Efficiency and Productivity…………………………….. 106

4.1 T. reesei Cellulases: The Current Industry Standard………………………………………. 106

4.2 Searching for Synergy………………………………………………………………………………… 107

4.2.1 P-Glucosidase………………………………………………………………………………………….. 108

4.2.2 Glycosyl Hydrolase Family 61 109

4.2.3 Synergistic Hemicellulases………………………………………………………………………… 111

5 Producing Enzymes Economically………………………………………………………………. 115

5.1 Reduced Enzyme Recovery………………………………………………………………………… 117

6 Conclusions………………………………………………………………………………………………. 118

References ……………………………………………………………………………………………………. 118

Abstract Enzymes play a critical role in the conversion of lignocellulosic waste into fu­els and chemicals, but the high cost of these enzymes presents a significant barrier to commercialization. In the simplest terms, the cost is a function of the large amount of enzyme protein required to break down polymeric sugars in cellulose and hemicellulose to fermentable monomers. In the past 6 years, significant effort has been expended to re­duce the cost by focusing on improving the efficiency of known enzymes, identification of new, more active enzymes, creating enzyme mixes optimized for selected pretreated substrates, and minimization of enzyme production costs. Here we describe advances in enzyme technology for use in the production of biofuels and the challenges that remain.

Keywords Biomass • Enzymes • Hydrolysis

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Introduction

The utilization of lignocellulose for the production of fuels and chemicals has the potential to change the world economically, socially, and environ­mentally. Today roughly 87% of the energy used in the world is derived from non-renewable sources such as oil, natural gas, and coal, with total en­ergy consumption increasing at approximately 4% per annum. About 28% of that energy consumption is in the form of liquid transportation fuels, de­rived almost entirely from petroleum [1]. The long-term cost of continued use of these finite fuel sources can already be seen in increased conflict over their control and distribution, climate change linked to increased greenhouse gas emissions, and increasing prices, all of which negatively impact people around the world every day. Lignocellulosic biomass, in the form of plant materials such as grasses, woods, and crop residues, offers a renewable, geo­graphically distributed, greenhouse-gas neutral source of sugars that can be converted to ethanol or other liquid fuels via microbial fermentation.

In the past 30 years, ethanol produced from corn starch and sugarcane has been established as an economically viable supplement to gasoline. In the USA over the past 5 years, production has increased from 175 million gallons per year to nearly 4.5 billion gallons last year, and is growing at more than 25% per year. In the near future, the use of sugar and starch as feedstocks for fuel production is expected to face increasing competition with their direct use as food and animal feed, impacting both availability and price. Current estimates suggest that in the USA, starch-based ethanol output will reach a maximum of between 12 and 15 billion gallons per year [2]. To significantly impact the use of petroleum in the USA, which uses approximately 140 bil­lion gallons of gasoline per year, additional sources of fermentable sugar for ethanol production will be required.

Lignocellulosic biomass has the potential to become a major source of these fermentable sugars in the future. It is estimated that in the USA alone, more than one billion tons per year of biomass could be sustainably harvested in the form of crop and forestry residues, replacing as much as 30% of the total US gasoline consumption [3].

To turn the prospect of replacing a significant proportion of the current liquid fuels into reality, the conversion of lignocellulose to ethanol must be­come less expensive in both operating cost and capital investment. Current estimates suggest that the cost of producing cellulosic ethanol is $1.80/gal — lon or higher, or almost twice as high as the cost of producing ethanol from starch [4]. Part of this high cost results from a significantly higher esti­mated capital investment for the construction of cellulosic plants compared to starch-based production facilities. Cellulose-to-ethanol plants in current design scenarios require more unit operations, must be larger to accom­modate more dilute sugar streams, and in some cases require acid-resistant construction materials, which in sum are projected to increase the invest­ment more than fourfold relative to current dry milling starch-based ethanol plants (from $1.10/gallon installed capacity to $4.70/gal) [4]. On the operat­ing cost side, equipment replacement may be more frequent due to processing materials that are more abrasive than seed, enzyme cost will be significantly higher due to the increased complexity of the substrate and higher enzyme dosage required to release the sugars, and higher water consumption may be required to remove compounds that interfere with the hydrolysis and fermen­tation processes.

Starch is present in plants as an energy source for growing seeds, while lignocellulose is present as a structural cell wall component to give the plant rigidity; therefore it should be no surprise that the latter is much more resis­tant to enzymatic attack. On a protein weight basis, it takes anywhere from 40-100 times more enzyme to break down cellulose than starch, yet the cost of enzyme production is not substantially different (Novozymes, unpublished data).

In 2001, Novozymes was awarded a research subcontract by the US Depart­ment of Energy with the goal of reducing the cost of cellulases for ethanol production from biomass. This effort, called the Cellulase Cost Reduction Project, was administered by the National Renewable Energy Laboratory (NREL), with Novozymes providing expertise for enzyme improvement and production, and NREL contributing expertise in biomass pretreatment and enzyme evaluation. The stated goal of the project was to achieve a tenfold reduction in the cost of enzymes for the conversion of acid pretreated corn stover to ethanol in laboratory-scale testing. At the beginning of this work, the cost of providing a commercial cellulase preparation for the conversion of 80% of the cellulose in acid pretreated corn stover to fermentable glucose was estimated to be $5.40/gallon ethanol produced. During the course of the contract, significant advances were made in improving the efficiency of the cellulases, increasing the yield in production, and reducing the cost of pro­duction. In addition, work focusing on other enzyme activities required for effective enzymatic hydrolysis of lignocellulosic substrates other than acid pretreated corn stover was successfully conducted. In this manuscript, we highlight some of those efforts that have contributed to making enzymes for lignocellulose hydrolysis more affordable.

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